Microstructured haptotaxic implant

ABSTRACT

The invention relates to the field of tissue engineering and regenerative medicine, and particularly to a three-dimensional biomimetic tissue scaffold that exploits the use of three-dimensional print technology. Surface energy is controlled by precisely placing polymers with differing surface chemistry, and using surface texture and bulk composition to pattern absorbable and non-absorbable polymers for the purpose of promoting functional healing in a mammalian body.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/236,738, filed Apr. 21, 2022, which is a continuation of U.S. PatentApplication Ser. No. 15/809,766 filed Nov. 10, 2017, now U.S. Pat. No.11,013,827, which is a continuation of PCT Application No.PCT/US2017/003046, filed May 1, 2017, which claims the benefit ofpriority to U.S. Provisional Application No. 62/330,104, filed on Apr.30, 2016, the contents of which are hereby incorporated by reference intheir entirety.

TECHNICAL FIELD

The present disclosure provides in part engineered biomaterials,including a patterned biomaterial having organized fibers or dropscapable of acting as a synthetic extracellular matrix for promotingwound repair.

BACKGROUND OF THE INVENTION

The stiffness and elasticity of extracellular matrix in living tissuehas important implications in cell migration, gene expression, anddifferentiation. More precisely, microscopic variations in stiffness andelasticity appear to be an important differentiating property of naturaland synthetic tissue scaffolds. Cells actively sense extracellularmatrix rigidity and migrate preferentially towards stiffer surfaces in aphenomenon called durotaxis. They also detect elasticity and adjusttheir gene expression accordingly which has increasingly become asubject of research because of its impact on differentiation andfunctional wound healing.

Lo and colleagues formulated the hypothesis that individual cells candetect substrate stiffness by a process of active tactile exploration inwhich cells exert contractile forces and measure the resultingdeformation in the substrate.

We have discovered that surface energy or hydrophobicity, or moreprecisely the microscopic variation of surface energy, is the underlyingdriver of durotaxis. High stiffness and low elasticity is correlatedwith high surface energy at microscopic scale. The elasticity sensed bycells is a combination of the Young's modulus of the substrate and therigidity of the cellular attachment. Since the tensile strength of thecellular attachment is strongly affected by the surface energy of thesubstrate, we have found that surface energy is important in cellmotility and functionality. The stiffness of the substrate is importantin the equilibrium achieved by the factors of ⋅1) deformation of thesubstrate, 2) strength of cell attachment, and 3) the resultingdeformation of the cell shape due to factors Cl) and (2).

In the body, microscopic differences in rigidity and hydrophobicitywithin the extracellular matrix is a result of the qualitative andquantitative biochemical properties of the extracellular matrix. Inparticular, the concentration, distribution and categories of thevarious macromolecules that form the extracellular matrix meshwork.Though the extracellular matrix is composed of manyintracellularly-synthesized components—including a number ofglycosaminoglycans and fibrous proteins such as fibronectin, laminin,collagen, and elastin—it is the latter two fibrous proteins that aremost influential in defining the mechanical properties of naturalextracellular matrix.

The mechanical behavior of materials at the microscopic scale is oftendifferent from that at macroscopic scale. At the microscopic scalesurface effects may control the deformation properties due to theincreasing surface to volume ratio where surface effects becomepredominant and can significantly modify the macroscopic properties ofcellular dynamics.

Both elastin and collagen are proteins rich in hydrophobic amino acidssuch as glycine and proline, which form mobile hydrophobic regionsbounded by crosslinks between lysine residues. Differences in thehydrophobicity of collagen compared to elastin contributes to elastinbeing the elastic element in extracellular matrix and collagen being thestiffening element in extracellular matrix.

These two constituents exist in alternating fashion in extracellularmatrix. These observations have led to the present novel disclosure of asynthetic extracellular matrix mimic or patterned biomaterial that isstructurally and functionally different from existing synthetic tissuescaffolds and soft tissue mesh. These novelty and utility of the presentpatterned biomaterial will be understood more completely in view of thefollowing background material.

Cell adhesion is the binding of a cell to a surface or substrate, suchas extracellular matrix. Adhesion occurs from the action of proteins,called cell adhesion molecules, or sometimes adhesins. Examples of theseproteins include selectins, integrins, and cadherins. The action ofthese proteins is highly controlled by the surface energy orhydrophobicity of a surface. Cellular adhesion is essential in cellmotility and wound healing.

Adhesion occurs by reversible reactions which occur on cell surfaceproteins which are triggered by variations in surface energy. Forces andinteractions may include hydrolysis/hydrophobic reactions, electrostaticreactions, Brownian motion, and facilitation by polysaccharides orbiofilm polymers.

We have surprisingly found that the spatial frequency of adjacentsurfaces is important in facilitating lamellipodia attachment, cellularpolarization, and ultimately cellular motility. This finding isconsistent with the structure of extracellular matrix.

The animal extracellular matrix includes the interstitial matrix whichis hydrophilic relative to the basement membrane. Interstitial matrix ispresent between various animal cells in the intercellular spaces. Gelsof polysaccharides and fibrous proteins fill the interstitial space andact as an orienting environment relative to the basement membrane.Basement membranes are sheet-like depositions of hyaluronic add on whichvarious epithelial cells rest. Hyaluronic acid (Hyaluronan) is apolysaccharide consisting of alternating residues on D-glucuronic acidand N-acetylglucosamine, which also differ in their hydrophilicity.Hyaluronic acid in bulk is relatively hydrophobic and does not readilydissolve in water. It is not found as a hydrophilic proteoglycan.Hyaluronan is a polymer of disaccharides, themselves composed ofD-glucuronic acid and D-N-acetylglucosamine, linked via glycosidicbonds. Hyaluronan can be 25,000 disaccharide repeats in length.

On the other hand, proteoglycans have a net negative charge thatattracts positively charged sodium ions, which attracts water moleculesvia osmosis, keeping the ECM and resident cells hydrated. Proteoglycansmay also help to trap and store growth factors within the ECM. Thus, itcan be seen on theoretical grounds that a hydrophobic polarizingstructure surrounded by a hydrophilic environment, or more simply analternating hydrophilic/hydrophobic structure (as represented by themolecular structure of Hyaluronan), is important in cell motility.

Haptotaxis is the directional motility or outgrowth of cells, usually upa gradient of high surface energy cellular adhesion sites or substratebound chemoattractants. Haptotaxis is distinguished from chemotaxis inthat the chemoattraction is being expressed or bound on a surface,rather than the gradient in a fluid. In order for cells to migrate, theouter cell membrane needs to be directed or polarized, otherwise thecell membrane cannot develop a leading and trailing orientation.

We have discovered that alternating regions ofhydrophobicity/hydrophilicity is important in haptotaxis. Further, onedistinguishing feature between living extracellular matrix and synthetictissue scaffolds is the presence of haptotaxis. Haptotaxic gradients arenaturally present in the extracellular matrix of the body duringprocesses such as angiogenesis. Researchers have attempted to mimicextracellular matrix by designing biomaterials where gradients areestablished by altering the concentration of adhesion sites on a polymersubstrate. However, discretizing adhesion sites does not provide thepolarization needed to promote cellular motility.

In wound repair, cells move through the extracellular matrix vialamellipodia. The lamellipodium is a cytoskeletal protein actinprojection formed at a polarized edge of a cell. It contains aquasi-two-dimensional actin mesh; the whole structure propels the cellacross a substrate. Lamellipodia are a characteristic feature at thefront, leading edge, of motile cells. They are believed to be the actualmotor which pulls the cell forward during the process of cell migration.The tip of the lamellipodium is the site where exocytosis occurs inmigrating mammalian cells as part of their clathrin-mediated endocyticcycle. This, together with actin-polymerization promoted by a highenergy surface, helps extend the lamella forward and thus advance thecell's front. It thus acts as a steering device for cells.

Cell polarity arises primarily through the localization of specificproteins to specific areas of the cell membrane. Cell polarizationaffects cell shape and cell functions, including proliferation,differentiation, apoptosis, and motility [O'Neill et al., 1986; Singhviet al., 1994; Chen et al., 1997; Baill et al., 1998; Dike et al., 1999].For example, cells can be switched between growth and death programs byvarying the size and spatial frequency of discretized attachment [Chenet al., 1997]. In general, total cellular mass increases as cellspreading is promoted, whereas apoptosis is observed in cells where cellspreading is discouraged. Based on the observation that cell growth iscorrelated more closely with the extent of cellular spreading than thetotal area of cellular adhesion, it seems likely cellular polarizationis more important to cell motility than providing a featureless surfaceon which cells can attach and live.

Strategies for regenerating tissue usually involve promoting some aspectof cellular function, such as motility and differentiation. Unlike bloodor bone marrow tissues which can be regenerated by intravenous injectionof cells, regeneration of most tissues requires a template to guidetheir growth. Two-dimensional patterns are weak representations of thereal cell environment and therefore there is great need to createstructures or scaffolds that exhibit similar spatial control ofbioactive molecules as those of 2D surfaces but within a 3D geometry.The extracellular matrix found in living tissues is a complex 3D highlyhydrated environment made from many elements such as soluble or surfacebound molecules, proteins, enzymes, and physical cues like pores andtopographies. The precise spatial location of these molecules isstrongly affected by the regions of hydrophobicity within theextracellular matrix.

Another important consideration in developing a tissue repair scaffoldis the promotion of directed cell motility that leads to the formationof new blood vessels. Although some tissues can function with lowercapillary densities, adequate perfusion of metabolically active tissuerequires intimate localization of parenchymal cells to a densevasculature in a highly organized manner. For example, the liver has aprecisely defined organization in which hepatocytes and microvessels areinterdigitated in a highly aligned microarchitecture. In many cases, thedegree of order in healing tissue correlates with its functionality. Inaddition, the architecture of the vasculature itself, e.g., thebranching frequency and angles, alignment of vessels, and tortuosity,determines gradients of metabolite exchange and the overall flow fieldsthrough the tissue. Therefore, the engineering of such tissues canrequire approaches to define the geometric architecture of vascularnetworks for tissue-specific applications.

Certain cell-based pre-vascularization strategies of engineered tissueshave utilized randomly seeded cells embedded within a three-dimensionalmatrix. For example, investigators discovered that the speed ofvascularization can be increased by allowing endothelial cells to formrudimentary networks in vitro prior to implantation. It has beendemonstrated that implantation of scaffolds pre-seeded with endothelialcells facilitates tubulogenesis (the formation of interconnectedweb-like networks of interconnected endothelial cells) within thescaffold and eventual anastomosis (connection) of the newly formedtubules to host vessels within days.

Unfortunately, such networks are randomly distributed rather thandirected. And as described above, the formation of directed complextissue structures starts with directed cell migration. To date it hasbeen difficult to control the formation and structure of vessels in afixed and reproducible manner. For example, the random organization ofendothelial networks provides no directional guidance to incoming hostvessels, often resulting in only an outer shell of the implant becomesperfused, leaving the interior core under-perfused. Furthermore, thestrict spatial organization of cells, the surrounding extracellularmatrix, and vasculature can impact paracrine signaling gradients thatdefine cellular phenotypes and tissue function.

Lastly, surface energy of bulk-scale (>1 mm) materials is a function ofthree hierarchical structure scales: 1) the fine-scale (<1 micron)molecular structure of a substance, 2) the meso-scale (<50 micron)geometry of the substance surface and 3) the macro-scale (>50 micron)distribution of molecular types (e.g., hydrophilic vs hydrophobic).

The meso-scale is achieved by placing a texture on a surface such as afiber, membrane, or particle. The phenomenon is known variously assuperhydrophobicity, the Lotus effect, and the petal effect. By placingmeso-scale shapes, usually the superposition of multiple shapes ofdifferent size, can alter the surface energy of a substanceindependently of its molecular composition. This is an importantconsideration in applications which would benefit from decoupling theelasticity and stiffness, generally determined by the molecularstructure, from the surface energy or hydrophobicity. These texturedmodifications of a substance provides a second degree of freedom.

It would be advantageous to construct specific structures frombiocompatible synthetic or natural polymers, inorganic materials, orcomposites of inorganic materials with polymers, where the resultingstructure has defined pore sizes, shapes and orientations, particularlydifferent pore sizes and orientations within the same device, with morethan one surface chemistry, surface energy or texture at differentspecified sites within the device.

BRIEF SUMMARY OF THE INVENTION

The present disclosure introduces an additional third degree of freedom(macro-scale) and combines the molecular freedom of polymer science withthe superhydrophobicity freedom of surface science with the placementfreedom of substances and textures using three dimensional printingtechnology. Without being bound by theory, it is believed that combiningall three of these sciences can achieve a synthetic extracellular matrixmimic that capable of directing the proliferation, differentiation andfunctionalization of cells involved in wound repair.

It is therefore an object of the present invention to provide complexthree-dimensional spatial patterns for use in devices of novel designand composition for use in tissue regeneration.

It is another object of the present invention to provide designs andcompositions for making complex medical devices of bioerodible ornon-bioerodible materials or composites for either cell transplantationor matrix-guided tissue regeneration.

It is a further object of the present invention to provide tissuescaffolds that are the result of high precision printing of implantablemedical devices capable of directing cell proliferation,differentiation, apoptosis, and motility.

It is a still further an object of the present invention to producedevices which can selectively encourage the growth of one tissue typeover another at specific sites within the matrix by virtue of control ofsurface chemistry, surface energy, texture or release of growth factorat an intended region of the matrix.

In some embodiments, the present disclosure provides a three-dimensionalmatrix for tissue regeneration comprising at least three successivelayers of at least two biocompatible polymers, the layers arranged toform walls with open voids therebetween, the voids suitable for seedingor ingrowth of cells, the layers comprising a first polymer and a secondpolymer, and wherein the first and second polymers are disposed in analternating pattern along at least one axis of the matrix. In someembodiments, the polymers are solid below a temperature of about 36° C.The matrices are useful in a variety of implantable medical devices suchas surgical meshes and implantable prosthetics.

The above and other aspects can be achieved as is now described. Aprinter or raster device is used to lay down small spheres or threads ofbiomaterials to build by layering a three-dimensional implantable tissuescaffold. The printer device may contain multiple print heads to deliverdifferent biomaterials during the scaffold construction. Thebiomaterials are delivered in liquid form either as a polymer dissolvedin a solvent or as a melt. The spatial precision of the biomaterialplacement may be enhanced by electrospinning methods. Electrospinninguses an electrical charge to direct and draw very fine (typically on themicro or nano scale) fibers from a liquid.

The present disclosure provides engineered biomaterials, includingpatterned biomaterials having organized fiber structures arranged topromote cell motility in a biomimetic extracellular matrix. In certainembodiments, the biomaterials are delivered as continuous fibers. Incertain embodiments, the biomaterials are delivered as discrete spheresor drops that combine in liquid form when juxtaposed. In certainembodiments, the patterned biomaterial can include a plurality ofdifferent fibers or drops. The patterned biomaterials solidify when theycool or when the solvent evaporates. In certain embodiments, highsurface energy biomaterials are organized in dusters or islands within anetwork of low surface energy biomaterials.

In certain embodiments, the high and low surface energy biomaterials arefibers that are layered in alternating fashion to build walls of mesh orporous structure. In certain embodiments, the patterned biomaterialincludes a synthetic extracellular matrix scaffold comprised of abioabsorbable material. In certain embodiments, the patternedbiomaterial can include natural extracellular matrix substances, forexample, hyaluronan.

In certain embodiments, the patterned biomaterial can include incombination one or more fibers and one or more dusters or islands ofbiomaterials.

In certain embodiments, the patterned biomaterial can be deposited on asmooth two-dimensional surface. In certain embodiments, the patternedbiomaterial can be deposited on a textured two-dimensional surface, suchthat when the biomaterial solidifies it acquires a surface texture. Thesurface texture may be hierarchical, for example, a biomimetic of rosepetal.

In certain embodiments, the surface texture may be superhydrophobicpossessing a low surface energy. The surface texture may be chosen toencourage certain types of cells to colonize the patterned biomaterialand discourage colonization by other cell types. For example, thesurface may encourage motility of endothelial cells and discourageattachment of bacterial cells. In certain embodiments, the surfacetexture may encourage mechanical integration of the patternedbiomaterial with living tissue by inhibiting migration of the patternedbiomaterial within a living body. In certain embodiments, one or morechemotaxic substances are embedded on or in the fibers or placed injuxtaposition with the patterned biomaterial. For example, thechemotaxic substances can include, but are not limited to, peptides,proteins, carbohydrates, collagen, fibrin, fibrinogen, matrigel,agarose, polyethylene glycol, dextran, hyaluronic acid, or a combinationthereof. In addition, the chemotaxic substance may be a naturalsubstance of botanical origin. For example, any of several constituentsof the genus Boswellia, in particular triterpenoids. Preferably, thenatural substances are made biocompatible by polymerization with apolyether compound.

In certain embodiments, the method of fabricating a patternedbiomaterial includes the placement of soluble fibers or drops whereinafter formation of the patterned biomaterial these soluble parts may beremoved by dissolution upon contact with a liquid and the insolubleparts remain.

In certain embodiments, channels can be formed in the patternedbiomaterial by dissolution. The channels can differ in shape, diameter,and length to generate cellular ingrowth of varying structure. Forexample, the patterned biomaterial may be entirely solid in the bulkvolume of which are disposed soluble cylindrical, bifurcating, Y-shaped,or branching structures. In certain embodiments, the soluble structurescan be fabricated into shapes that differ in length, diameter, anddensity. In certain embodiments, the properties of the solublestructures can be altered to suit a particular application. In certainembodiments, the overall network organization can be defined, forexample, by the number and location of branch points, connections,three-dimensional organization, degree of anisotropy, alignment,diameters, lengths, and more.

In certain embodiments, the combination of a bioabsorbable but insolubleportion with a soluble portion can result upon immersion in a liquid apatterned biomaterial comprising one or more wells and/or channels togenerate dusters or islands of colonizing cells. In certain embodiments,the islands and/or cluster of colonizing cells can be fabricated intostructures that differ in diameter, density, three-dimensionalorganization and shape. In certain embodiments, the properties of thedusters or islands of colonizing cells can be altered to suit aparticular application.

In certain embodiments, the patterned biomaterial can be used to treatan ischemic tissue of a subject. For example, the patterned biomaterialcan be implanted onto a subject to increase the blood flow to regions ofa tissue that are not receiving adequate blood flow. In certainembodiments, pattern biomaterial can be used to treat cardiac ischemia,peripheral vascular disease, or chronic wounds such as diabetic ulcers.

Examples of printing methods include stereo-lithography, selective lasersintering, ballistic particle manufacturing, fusion deposition modeling,directed electrospinning and three-dimensional printing. In a preferredembodiment, three-dimensional printing is used to precisely arrangealternating layers of high surface energy and low surface energy fibersin a biomimetic pattern designed to cause cell growth and proliferationin a synthetic matrix that mimics natural extracellular matrix. Forexample, three-dimensional printing can be used to create a porousbioerodible matrix having interconnected pores or channels, typicallybetween 0.5 and 5 mm, which are separated by walls approximately 100 to1000 microns thick, the walls constructed of fibers of alternatingsurface energy and possessing a diameter of approximately 5 to 100microns.

The macrostructure and porosity of the device can be manipulated bycontrolling printing parameters, the type of polymer and drop/fibersize, as well as the solvent and/or temperature. In the case of varyingthe solvent content or temperature, the degree of bonding between fibersor drops can be controlled. Porosity of the matrix walls, as well as thematrix as a whole, can be manipulated using printing methods. Structuralelements can be printed in the patterned biomaterial that maintain theintegrity of the matrix of an implantable device during resorption in abody. For example, to provide support, the porosity of the walls of thedevice can be filled with resorbable inorganic material, which canfurther provide a source of mineral for a repair site of a bone.

Alternatively, one or more of the materials used in the manufacture ofthe present patterned biomaterial may be a prepolymer in the process ofpolymerization or polymerizable by application of heat or light,especially UV light.

The use of a prepolymer capable of polymerization at a point ofapplication can form molecular structures different from polymerscapable of dissolving in a solvent or melting. In particular, polymerswith cross links do not melt and cannot be dissolved in a solvent.

BRIEF DESCRIPTION OF THE SEVERAL VIEW OF THE DRAWINGS

FIG. 1A-1D depicts several views of an exemplary rose surface patternmimic.

FIG. 2 depicts an exemplary square mesh prepared using an electrospindevice and computer aided design

FIG. 3A-3C depicts the construction of a chain mail patterned matrixmaterial.

FIG. 4 depicts a composite sheet and mesh patterned biomaterial 400.

FIG. 5 depicts a three-dimensional mesh 500 is depicted.

FIG. 6 depicts an exemplary mesh device as used with human tissue havinga defect to be repaired.

FIG. 7 is an image of a mesh according to the present disclosure havingalternating hydrophobic and hydrophilic fibers.

FIG. 8 is another image of an embodiment of a mesh according to thepresent disclosure.

FIG. 9 is a further design of a device, in which fibers are arranged ina series of stacked triangles. The polymer fibers can alternate betweenhydrophilic and hydrophobic fibers.

FIG. 10 depicts an alternative rose petal mimic having a hierarchicalgrid pattern with created by layers of varying width.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides engineered biomaterials, including apatterned biomaterial having organized fibers or drops capable of actingas a synthetic extracellular matrix for promoting wound repair.

As used herein, the term “patterned biomaterial” refers to solidnaturally-derived and/or synthetic substances that are organized intostructures that resemble cylinders, rods, strings, or filaments andnetworks of such structures.

A matrix of these biomaterials can lead to enhanced integration of thesematerials into host organisms, wherein host cells can invade orintegrate in a manner guided by the architecture and composition of thematrix. This integration can involve blood vessels, thus providing astrategy for enhancing the healing aspects of an implantable prostheticby allowing for increased vascularization of a wound site. Accordingly,these matrices are particularly useful in surgical applications formammalian subjects, and in particular, human subjects.

In addition, these patterned biomaterials provide a scaffold forpromoting angiogenesis in a host tissue. This integration can alsoinvolve other host systems such as the nervous system, muscle, bone, orimmune system, and thereby promote new tissue innervation, muscleintegration, bone integration, or immune surveillance, respectively. Incertain embodiments, a patterned biomaterial of the present disclosurecan include chemotaxic substances and additional naturally⋅-derivedand/or synthetic scaffolding.

In certain embodiments, the present disclosure provides a patternedbiomaterial that encourages cells to organize in dusters, layers and/orlines, in particular bifurcating lines. The bifurcating lines may have afractal dimension characteristic of capillary networks.

As used herein, the term cell “clusters” and/or “lines” refer to one ormore cells, with or without extracellular matrix that are organized instructures that resemble balls, discs, or islands when described asdusters or organized as interconnected filamentary networks whendescribed as lines.

The patterning of the biomaterial can be used in conjunction withbioactive or chemotaxic agents, such as paracrine factors. Thesebioactive agents act to modulate how host tissue and cells respond tothe patterned biomaterial. In certain embodiments, a duster or line ofthe present disclosure can include cells and a naturally-derived and/orsynthetic scaffolding of the present invention in combination.

In certain embodiments, the patterned biomaterial of the presentinvention is used to repair soft tissue during a surgical procedure.There is a need to encourage the formation of functional tissue asopposed to the usual formation of scar tissue. Functional tissue isassociated with fewer adverse events and is capable of self-repair. Scartissue serves a short-term function in joining tissue together, butgenerally is remodeled by the body. The remodeling process can result inthe surgical repair failing post-surgically, as is common in herniarepairs.

Functional tissue requires blood supply, unlike scar tissue which isgenerally sparsely populated with vessels or avascular. Accordingly,there is a need for a soft tissue repair device for inducingangiogenesis, the formation of new blood vessels, in a controlledfashion within organized three-dimensional tissue constructs. There isalso a need to induce blood vessel formation of a tissue in a subjectthat does not receive adequate blood flow. The present disclosureaddresses this need by engineering a patterned biomaterial that mimicsnatural extracellular matrix by providing a substrate on which cells aremotile. In this sense, the patterned biomaterial acts as a scaffold thatpossesses high surface energy and low surface energy regions thatgeometrically direct the formation of functional constructs of cells.

The patterned biomaterials of the present disclosure promote the rapidformation of vessels that are spatially delineated, providing novelapproaches to vascularizing issues formed by the body during woundrepair. These patterned biomaterials are also useful in treatingischemic diseases and promoting tissue healing and integration at sitesnormally undersupplied with blood.

Implantation of patterned biomaterial into a subject can lead toengraftment, remodeling of the local microenvironment, anastomosis, andformation of stable capillaries within an implanted scaffold thatdirects blood vessels and blood flow. By employing synthetic materialswith geometrically controlled distributions of dusters and lines of highand low surface energy, the subsequent formation of blood vessels invivo is able to be spatially controlled.

The arrangement of fibers of different surface energy into patternednetworks of the present disclosure provides a means to support rapidinvasion and integration of host vasculature into the device to generateperfused, functional blood vessels by providing a pre-specifiedarchitecture as a template in which the new blood vessels mirror thearchitecture of fibers of the implanted tissue scaffold. Thearchitecture of the fibers of a patterned biomaterial can also encouragecertain types of cellular infiltrates. For example, in certain casesendothelial cells are promoted and in other cases fibroblasts areencouraged, and in still another case the distribution and densities ofcombinations of cell types are promoted.

The material and geometry of the patterned biomaterial defines the invivo architecture of the blood vessels, connective tissue, muscle tissueand anchoring networks that are preferred in the post-operativefunctional healing of a wound site. Because these patterned networks actas templates for the formation of blood vessels, benefit the motility ofinvading host tissue, directs their rate of propagation and spatialdistribution they can rationally impact the rate and extent of host cellintegration, and thus be used as a means to direct revascularizationfrom a well perfused site to reach into and support ischemic tissues. Incertain embodiments, the pattern biomaterial is resorbable such that theoriginal matrix can be partially or entirely replaced by host cells andtissue, with the architecture of the patterned biomaterial beingtemplated and preserved by the new host tissue.

This use of patterning technology in combination with variations incomposition that determine rates of resorption and surface energy valuesis novel in that such a construct mimics the composition of naturalextracellular matrix. Accordingly, such a synthetic biomaterialpossesses some of the same functionality as extracellular matrixregarding the organization of infiltrating cells at an implant site forcontrolling the ultimate in vivo composition and distribution of tissuetypes. Functional promotion and patterning of cells and subsequentformation of vessels in vivo constitutes a significant technical advancewithin the field of surgical medicine.

In some embodiments, the present disclosure provides a three-dimensionalmatrix for tissue regeneration comprising at least three successivelayers of at least two biocompatible polymers, the layers arranged toform walls with open voids therebetween, the voids suitable for seedingor ingrowth of cells, the layers comprising a first polymer and a secondpolymer, and wherein the first and second polymers are disposed in analternating pattern along at least one axis of the matrix. The firstpolymer can be hydrophobic, and the second polymer can be hydrophilic.In other embodiments, the first polymer is lipophilic, and the secondpolymer is hydrophilic. The first and second polymers can be nanofibershaving a diameter ranging from 100 nm to 5 microns or 100 nm to 1 micronor 100 nm to 500 microns. Useful hydrophobic polymers include withoutlimitation polypropylene, polycaprolactone, and polylactic acid. Usefulhydrophilic polymers include without limitation polyether urethanes,polyester urethanes, or polyhyaluronic acid.

In some embodiments, the nanofibers are arranged as stacked layer suchthat they form wherein the walls that intersect at junctions alongparallel first axes, such as in a grid pattern. The first and secondpolymers may be disposed in an alternating pattern along the first axes,or they may be disposed in an alternating hydrophilic-hydrophobicpattern along a second axes that are perpendicular to the first axes.

The layers may further include surface pattern is provided on one ormore layers of said device.

The surface energy is advantageously affected by the alternatinghydrophobic-hydrophilic pattern. Additionally, the surface energy can beadvantageously be affected by including a surface pattern such that thesurface energy is varied on at least three spatial scales comprising 1)a macro scale ranging from 50 micron to 1 mm obtained by forming layersof polymers of different surface energy, 2) a meso scale ranging from 1micron to 50 micron obtained by placing a surface pattern on thepolymers, and 3) a fine-scale of less than 1 micron obtained byvariation of molecular structure.

The present disclosure further provides a three-dimensional medicaldevice comprising the matrices disclosed herein, wherein the matrix isformed on an implantable layer such that the final three-dimensionaldevice comprises the matrix joined to an implantable layer. In someembodiments, the layer and the matrix are bioabsorbable, and wherein thelayer bioabsorbs more rapidly than the matrix.

In another embodiment, a matrix for tissue regeneration comprises atleast three successive layers of at least two biocompatible polymers thelayers arranged as loops having open voids therebetween, the voidssuitable for seeding or ingrowth of cells, the layers comprising a firstpolymer and a second polymer, wherein the first and second polymers aredisposed as interlocking loops, and the first and second polymers arearranged in an alternating pattern. The first polymer can behydrophobic, and the second polymer can be hydrophilic. In otherembodiments, the first polymer is lipophilic, and the second polymer ishydrophilic. The first and second polymers can be nanofibers having adiameter ranging from 100 nm to 5 microns or 100 nm to 1 micron or 100nm to 500 microns. Useful hydrophobic polymers include withoutlimitation polypropylene, polycaprolactone, and polylactic acid. Usefulhydrophilic polymers include without limitation polyether urethanes,polyester urethanes, or polyhyaluronic acid.

The layers may further include surface pattern is provided on one ormore layers of said device.

The surface energy is advantageously affected by the alternatinghydrophobic-hydrophilic pattern. Additionally, the surface energy can beadvantageously be affected by including a surface pattern such that thesurface energy is varied on at least three spatial scales comprising 1)a macro scale ranging from 50 micron to 1 mm obtained by forming aportion of said loops from a polymer of a surface energy different fromthe surface energy of the remaining said loops formed from a secondpolymer, 2) a meso scale ranging from 1 micron to 50 micron obtained byplacing a surface pattern on the polymers, and 3) a fine-scale of lessthan 1 micron obtained by variation of molecular structure.

The aforementioned matrix may be formed on an implantable layer suchthat the final three-dimensional device comprises the matrix joined toan implantable layer. In some embodiments, the layer and the matrix arebioabsorbable, and wherein the layer bioabsorbs more rapidly than thematrix.

In another embodiment, a three-dimensional matrix for tissueregeneration comprises at least three successive layers of at least twobiocompatible, the layers arranged in a series of stacked triangles, thetriangles providing open voids, the voids suitable for seeding oringrowth of cells, the layers comprising a first polymer and a secondpolymer, and wherein the first and second polymers are disposed in analternating pattern along at least one axis of the matrix. The trianglesare in some embodiments superimposed on one another such that that thesuperimposition of triangles (here 9) does create various quadrangles,pentagons, and a hexagon. In some embodiments, the matrix comprises atleast nine layers of triangles of varying sized, and substantiallyreplicates a sri yantra pattern.

Three-Dimensional Printing

Suitable devices include both those with a continuous jet stream printhead and a drop-on-demand stream print head. In the former case, a lineof polymer is directed. In the second case, a drop of polymer isdirected. A high-speed printer of the continuous type, for example, isthe Dijit printer made and sold by Diconix, Inc., of Dayton, Ohio, whichhas a line printing bar containing approximately 1,500 jets which candeliver up to 60 million droplets per second in a continuous fashion andcan print at speeds up to 900 feet per minute.

Both raster and vector apparatuses can be used. A raster apparatus iswhere the print head goes back and forth across the bed with the jetturning on and off. This can have problems when the material is likelyto clog the jet upon settling. A vector apparatus is similar to an x-yprinter. Although potentially slower, the vector printer may yield amore uniform finish.

The object of three-dimensional printing is to create a solid-stateobject by ink-jet printing a binder into selected areas of sequentiallydeposited layers of powder. In the present disclosure, this process ismodified in that powder is not required. The drop or line that isinitially liquid becomes a volumetric solid when deposited on a surface.In this sense, the process is more like ink in an ink-jet printingprocess, where a third dimension is created by the creation ofsuccessive layer of deposited polymer.

Instructions for each layer can be derived directly from acomputer-aided design (CAD) representation of the patterned biomaterial.The area to be printed is obtained by computing the area of intersectionbetween the desired plane and the CAD representation of the object. Afirst layer is joined to a second layer by the liquid state of thepolymer being deposited during the time of creation of the second layer.The liquid state of the second layer partially melts or dissolves intothe first solid layer to form the three-dimensional structure insuccessive layers.

While the layers become hardened or at least partially hardened as eachof the layers is laid down, once the desired final biomaterialconfiguration is achieved and the layering process is complete, in someapplications it may be desirable that the form and its contents beheated or cured at a suitably selected temperature to further promotebinding of the discrete lines or drops.

Construction of a three-dimensional component by printing can be viewedas the knitting together of structural elements, e.g., drops or lines.These elements are called microstructural primitives. The dimensions ofthe primitives determine the length scale over which the microstructurecan be varied. Thus, the smallest region over which the surface energyof the patterned biomaterial can be varied has dimensions near that ofindividual microstructural primitives. Droplet primitives havedimensions that are very similar to the width of line primitives, thedifference is whether the material is laid down in a continuous line ordiscrete drops. The dimensions of the line primitive depend on thepolymer viscosity and surface tension. A line primitive of 10-micronwidth is in certain cases possible, more typically the dimension is40-60 microns. Higher print head velocities and lower polymer viscosityproduce finer lines.

When solvents are used, the drying rate is an important variable in theproduction of patterned biomaterials by three-dimensional printing. Veryrapid drying of the solvent tends to cause warping of the printedcomponent. Much, if not all, of the warping can be eliminated bychoosing a solvent with a low vapor pressure. For example, patternedbiomaterials prepared by printing with a solution of polymer andchloroform have nearly undetectable amounts of warpage, while largeparts made with methylene chloride exhibit significant warpage. It hasbeen found that it is often convenient to combine solvents to achieveminimal warping and adequate bonding between the particles. Thus, anaggressive solvent can be mixed in small proportions with a solvent withlower vapor pressure.

Ballistic Particle Manufacturing (BPM) and Fusion Deposition Modeling(FDM)

Ballistic particle manufacturing is in some respects more like thepresent methods of printing than the traditional powder-basedthree-dimensional printing method, although both can be adapted to themanufacture of the present patterned biomaterial. Ballistic particlemanufacturing uses an ink-jet printing apparatus wherein an ink-jetstream of liquid polymer or polymer composite material is used to createthree-dimensional objects under computer control, similar to the way anink-jet printer produces two-dimensional graphic printing.

A patterned biomaterial device is formed by printing successivecross-sections, one layer after another, to a target using a coldwelding or rapid solidification technique, which causes bonding betweenthe drops and the successive layers of drops.

Fusion deposition modeling employs an x-y plotter with a z-directionmotion to position an extrudable filament formed of a polymericmaterial, rendered fluid by heat or the presence of a solvent. Asuitable system is available from Stratasys, of Minneapolis, Minn.

Ballistic particle manufacturing, fusion deposition modeling andthree-dimensional printing are related in the sense that all threeapproaches control the deposition of matter in small areas. This aspectis advantageous in the present application to the extent that localcomposition can be specified and constructed for any desiredthree-dimensional profile. The composition control is only limited bythe resolution of the particular apparatus used for construction.

Fusion deposition modeling builds structures by extruding a finefilament of plastically deformable material through a small nozzle. Thenozzle is directed over the built surface by appropriate x, y and zmotion control so as to yield the desired three-dimensional structure.Similarly, ballistic particle manufacturing involves motion control ofan ink jet print head to deposit matter in the form of small droplets.Appropriate control of where the droplets are printed permits theconstruction of a desired three-dimensional shape. Three-dimensionalprinting uses two sources of material: the material that makes up theporous layer and the material that is printed. All three are adaptableto manufacture of the present patterned biomaterial, each with distinctadvantages regarding the chosen architecture biomaterial architecture.

Local composition control using fusion deposition modeling and ballisticparticle manufacturing requires the application of multiple printing orextrusion heads. A similar approach can be followed withthree-dimensional printing by using multiple print-heads. Alternatively,multiple droplets may be printed into the same location when usingthree-dimensional printing to increase the local composition of thespecies contained in the printed solution.

Selection of Polymers

The printing method of the patterned biomaterial relies on the polymerconstituents being in a liquid phase. The liquid phase is typicallyrealized by dissolution of a solid polymer in a solvent or by melting.In the case of a melt phase, it is preferable to select polymers havingrelatively low melting points, to avoid exposing resorbable polymers toelevated temperatures. Resorbable polymers are typically susceptible tothermal degradation.

A number of polymers are commonly used in the construction ofimplantable medical devices. Unless otherwise specified, the term“polymer” will be used to include any of the materials used to form thepatterned biomaterial matrix, including polymers and monomers which canbe polymerized or adhered at point of application to form an integralunit.

In a preferred embodiment the microstructural elements are formed of apolymer, such as a synthetic thermoplastic polymer, for example,ethylene vinyl acetate, poly(anhydrides), polyorthoesters, polymers oflactic acid and glycolic acid and other a hydroxy acids, andpolyphosphazenes, a protein polymer, for example, albumin or collagen,or a polysaccharide containing sugar units such as lactose.

In a more preferred embodiment the polymers are absorbable polyurethanecontaining lactide diol blocks capable of resorbing in vivo. The lactidediol blocks are linked with ethylene diols and/or propylene diols viaurethane or urea links. By varying the proportion of ethylene diol topropylene diol, as well as the choice of the linking diisocyanate, thesurface energy of the resulting polymer can be modified to achieve adesired specification. Generally, these molecules are called polyesterpolyurethanes or polyesterurethanes.

An example of a polyesterurethane is an aliphatic polyester basedpolyesterurethane consisting of poly(l-lactic add) and poly(ethylenesuccinate) prepared via chain-extension reaction of poly(l-lacticacid)-diol and poly(ethylene succinate)-diol using 1,6-hexamethlyenediisocyanate as a chain extender. The poly(l-lactic add)-diol issynthesized by direct polycondensation of I-lactic acid in the presenceof 1,4-butanediol.

Poly(ethylene succinate)-diol can be synthesized by condensationpolymerization of succinic acid with excessive ethylene glycol.

The polymer can be non-biodegradable or biodegradable, typically viahydrolysis or enzymatic cleavage. In the case of polymers for use inmaking devices for cell attachment and growth, polymers are selectedbased on the ability of the polymer to elicit the appropriate biologicalresponse from cells, for example, attachment, migration, proliferationand gene expression.

An alternative material is a polyester in the polylactide/polyglycolidefamily. These polymers have received a great deal of attention in thedrug delivery and tissue regeneration areas for a number of reasons.They have been in use for over 30 years in surgical sutures, are Foodand Drug Administration (FDA)-approved and have a long and favorableclinical record. A wide range of physical properties and degradationtimes can be achieved by varying the monomer ratios in lactide/glycolidecopolymers: poly-L-lactic acid and poly-glycolic acid exhibit a highdegree of crystallinity and degrade relatively slowly into shards.Copolymers of poly-L-lactic acid and Poly-glycolic acid are amorphousand rapidly degraded into a gel state. The advantage of thepolyesterurethane polymers over the polyester polymers is that theformer degrade both into a gel state and are true surface-erodingpolymer. As a consequence, polyesterurethanes have a preferreddegradation state while retaining for a longer period the originalpatterns of the biomaterial. However, there are applications where eachare preferred.

In some embodiments, non-polymeric materials can also be used to formthe matrix either alone or in combination with a polymer. Examplesinclude organic and inorganic materials such as hydoxyapatite,bone-derived apatite, calcium carbonate, other bone substituting agents,buffering agents, and lactose, as well as other common excipients usedin drugs, which are solidified by application of adhesive rather thansolvent.

The selection of the solvent for chemotaxic agents delivered on aresorbable polymer matrix depends on the desired mode of release of thechemotaxic agent. In the case of a totally resorbable device, a solventis selected to deliver the chemotaxic agent alone and when delivereddissolves the deposited polymer matrix or is selected to contain asecond polymer which is deposited along with the chemotaxic agent.

In the first case, the printed chemotaxic droplet locally dissolves theunderlying polymer matrix and begins to evaporate and thus is adherentto the surface of the immediate underlying polymer matrix layer. In thesecond case, the drug is effectively deposited in a second polymermatrix after evaporation since the dissolved polymer is deposited alongwith the chemotaxic agent. The first case releases the chemotaxic agentrapidly and creates the highest concentration gradient when placed invivo. The second case releases the chemotaxic agent more slowly sincerelease depends in part of the resorption of the carrier polymer. Inthis second case, the concentration of chemotaxic agent is more uniformand constant over time.

The solvent evaporation rate is primarily determined by the vaporpressure of the solvent. There is a range from one extreme over whichthe polymer is very soluble, for example, 30 weight percent solubility,which allows the polymer to dissolve very quickly, during the timerequired to print one layer, as compared with lower solubilities. Thedegree to which prior layers are dissolved during application of asubsequent layer depends on the solubility of the polymer in thesolvent. Fine fibers are more completely dissolved than fibers withlarger diameters.

Polymer Concentration

In general, microstructural element are a resorbable polymer such aspolyesterurethane or polyester of molecular weight 5,000-200,000, in asolvent such as chloroform or a mixture of chloroform and aless-volatile solvent such as ethyl acetate to minimize warping. Thesurface energy of these can be varied by varying the proportion ofhydrophilic and hydrophobic blocks in the polymer. Alternatively, adifferent polymer may be used such as poly-lactic acid, poly-glycolicacid or polycaprolactone.

The polymer concentration in a microstructural element solution willgenerally be at the limit of what can be accommodated by the nozzle,both to maximize the amount of solid polymer delivered and to minimizemigration of the solvent away from the point of application in theformation of a patterned biomaterial. Reduced solvent migrationincreases the resolution of the microstructural elements of priordeposited layers, e.g., reduces swelling or geometrical slumping.

The upper limit of polymer concentration is 15% for poly-L-lactic acidof 100,000 MW. This concentration of polymer may in some cases makeprinting of commercially viable devices impossible. The cases where thepolymer is sparingly soluble, a filler may be used. Microstructuralelement volume can be increased by including small crosslinked orotherwise less soluble particles in the printing solution.

For example, polyglycolic acid is not soluble in chloroform or ethylacetate. Nanoparticles of crosslinked polyesterurethane can be includedin the printing solution (particles up to microns in diameter can beaccommodated through most nozzles) to increase the polymer content whichis printed.

The amount of matter which is printed into the biomaterial can also beincreased by including small inorganic particles in the polymersolution, for example, bone derived apatite.

Surface Texture Considerations

The manner in which the microstructural elements are laid downdetermines a surface texture between these elements. There is a firstdistinction in surface texture achieved by the dimensions of themicrostructural elements. There are two principal modes: a dropletconfiguration and a line configuration. Drops are discrete in threedimensions, whereas lines are discrete in two dimensions.

In the droplet mode the drops can be spaced apart in on a surface, andthey can be joined together by a subsequent layer of drops in staggeredform or joined together by a line. The drops can be spaced closertogether to slightly touch, creating an undulating profile, or they canbe placed in dose proximity so that they effective merge beforesolidifying. In the creation of islands, they can be stacked in pyramidfashion in a vertical direction.

In the line mode the lines are generally laid down in alignment with theprevious line. However draping configurations can be achieved. Forexample, a partial wall can be formed of several aligned lines on top ofwhich a line is placed such that it crosses this partial wall inundulatory fashion, such that adhesion between the wall and the line isonly at points. After solidification, these draping features typicallyare free to move away from the established wall structure. Drapingfeatures can be placed at points intermediate during the formation of acomplete wall. In addition, a partial wall can be fenestrated bysubsequent layers of droplets built up to form the edges of windows, thetop edge of which is dosed by the subsequent addition of lines. Theselines typically will droop down into the established fenestrations. Byvarying the deposit speed of the final lines one can create amultiplicity of drooping lines into the fenestration of differentlengths creating a curtain of drooping lines.

Alternatively the microstructural elements can be deposited on a planewith a mold pattern. For example, the mold pattern can be asuperhydrophobic pattern capable of generating a Wenzel-Cassie effect ora Wenzel-Baxter effect. Other surface textures can be achieved byincorporating on or in deposited microstructural element a variety ofsolid particulate. The solid particulate may be a permanentnanostructure, such as a nanotubule, a bucky ball, or any of variouslyknown nanoparticulate geometries. The solid particulate may be soluble,such that when the patterned biomaterial is placed in a solvent theparticulate are partially or entirely removed without affecting theremaining portion of the biomaterial.

In addition, directed and random writing techniques can be combined. Forexample, at various points during the construction of a directedstructure a spray or electrospinning technique could be employed todeposit randomly oriented fibrous or particulate masses.

Bioactive Agents

There are essentially no limitations on the bioactive agents that can beincorporated into the patterned biomaterials, although those agentswhich produce a chemotaxic effect are most desirable in wound healing ortissue scaffolding applications. Bioactive agents need not beincorporated as a liquid, they can be processed into particles usingspray drying, atomization, grinding, or other standard methodology, orthose agents which can be formed into emulsifications, microparticles,liposomes, or other small particles, and which remain stable chemicallyand retain biological activity in a polymeric matrix, are useful.

Examples of chemotaxic agents generally include proteins and peptides,nucleic acids, polysaccharides, nucleic acids, lipids, and non-proteinorganic and inorganic compounds. Examples of other bioactive agents havebiological effects including, but not limited to, anti-inflammatories,antimicrobials, anti-cancer, antivirals, hormones, antioxidants, channelblockers, and vaccines. It is also possible to incorporate materials notexerting a biological effect such as air, radiopaque materials such asbarium, or other imaging agents.

In a preferred embodiment for tissue regeneration matrices, cell growth,differentiation, and/or migration modulators are incorporated intospecific regions of the device at the same level of resolution as thepores and channels. These may act in combination with surface texture,surface energy, and overall shape and distribution of themicrostructural elements to achieve an extracellular matrix mimic withcontrollable tissue directing functionality.

Of particular interest are surface-active agents which promote celladhesion, such as an RGD peptide, or a material which inhibits celladhesion, such as a surfactant, for example, polyethylene glycol or aPluronic (polypropylene oxide-polyethylene oxide block copolymers).

For example, it may be desirable to incorporate adhesion peptides suchas the RGD adhesion peptide into certain channels (e.g., those for bloodvessel ingrowth). An adhesion peptide, such as the peptide having ahydrophobic tail marketed by Telios (La Hoya, Calif.) as Peptite, can bedissolved in water and deposited onto the surfaces of pores in thepatterned biomaterial.

The surface can be modified to prevent cellular adhesion. This may bedesirable to prevent excessive soft connective tissue ingrowth into thedevice from the surrounding tissue, and can be accomplished, forexample, by depositing an aqueous solution of a pluronic or poloxamer inthe voids. The hydrophobic block of such copolymers will adsorb to thesurface of the channels, with the hydrophilic block extending into theaqueous phase. Surfaces with adsorbed pluronics resist adsorption ofproteins and other biological macromolecules.

In certain embodiments, the patterned biomaterial can contain one ormore of bioactive substance(s) including, but are not limited to,hormones, neurotransmitters, growth factors, hormone, neurotransmitteror growth factor receptors, interferons, interleukins, chemokines,cytokines, colony stimulating factors, chemotactic factors,extracellular matrix components, and adhesion molecules, ligands andpeptides; such as growth hormone, parathyroid hormone (PTH), bonemorphogenetic protein (BMP), transforming growth factor-alpha(TGF-alpha), TGF-beta1, TGF-beta2, fibroblast growth factor (FGF),granulocyte/macrophage colony stimulating factor (GMCSF), epidermalgrowth factor {EGF), platelet derived growth factor (PDGF), insulin-likegrowth factor (IGF), scatter factor/hepatocyte growth factor (HGF),fibrin, collagen, fibronectin, vitronectin, hyaluronic acid, anRGD-containing peptide or polypeptide, an angiopoietin and vascularendothelial cell growth factor (VEGF). For example, the patternedbiomaterial can include a biologically effective amount of VEGF.

Porosity

Porosity is a void in the patterned biomaterial that bridges two sidesof the device. Porosity of printed biomaterials can be created either atthe level of the microstructural element size or at a macroscopic size(>1 mm). At the level of the microstructural size, porosity iscontrolled by where the elements are placed, and thus pore size andshape can vary in three dimensions.

Porosity at a sub-element size level can be created in a variety ofways. Printing a polymer solution onto a bed of fibers which are notsoluble in the polymer solution and too large in one dimension to beprinted and which can be subsequently dissolved with a solvent thatdoesn't affect the polymer can create pores. Alternatively, the polymersolution can be deposited onto a bed containing a foaming agent.Alternatively, the polymer solution can be deposited on a heated bedthat caused the solvent to pass into the gaseous phase before leavingthe polymer, thus creating gaseous voids in the polymer, some of whichmay be inter-connected.

Patterned Biomaterials Comprising Living Cells

In certain embodiments, the patterned biomaterial can be seeded withcells. In certain embodiments, the patterned biomaterial can becomprised of one or more cell types. Cells can confer tissuefunctionality and provide structures, which can replace or facilitatethe repair of a tissue of the subject. For example, the patternedbiomaterial can include, but is not limited to, muscle cells to providecontractile structures, vascular and/or neural cells to provideconductive elements, metabolically active secretory cells, such as livercells, hormone synthesizing cells, sebaceous cells, pancreatic isletcells or adrenal cortex cells to provide secretory structures, stemcells, such as bone marrow-derived or embryonic stem cells, dermalfibroblasts, in keratinocytes, Schwann cells for nerve implants, smoothmuscle cells and endothelial cells for vessel structures, urothelial andsmooth muscle cells for bladder/urethra structures and osteocytes,chondrocytes, and tendon cells for bone and tendon structures, or acombination thereof. In certain embodiments, the patterned biomaterialcan include other cell types including, but not limited, to hepatocytesand chondrocytes.

Cells suitable for inclusion in the patterned biomaterial of the presentdisclosure can be derived from any suitable source. The subject, whichmay be a mammalian subject, particularly a human subject, to receive theimplant of the patterned biomaterial of the present disclosure candetermine the source of the cells to be included in the patternedbiomaterial. In certain embodiments, the cells can be derived from anautologous source. For example, the cells can be derived from thesubject to be implanted with the patterned biomaterial. For example,epithelial cells can be derived from the skin of the subject to beimplanted with the patterned biomaterial. In certain embodiments, thecells can also be generated from stem cells derived from various sourcesthat are then differentiated into the desired cell type. For example,the stem cells can be derived from the subject to be implanted with thepatterned biomaterial. In certain embodiments, cells can be cultured fora period of time under various conditions to induce certain phenotypesbefore placing the cells in the patterned biomaterial.

Applications Using the Patterned Biomaterials

In certain embodiments, the patterned biomaterial of the presentdisclosure can be implanted in a human subject. For example, in certainembodiments, the patterned biomaterial of the present disclosure can beimplanted in a subject by suturing the patterned biomaterial to fat padsor muscle tissue in the lower abdomen.

In certain embodiments, the patterned biomaterial of the presentdisclosure can be used to enhance vascularization in ischemic settings,such as, by acting as an angiogenic tissue scaffold to promoteneovascularization and ultimately increase blood flow to regions oftissues that are not receiving sufficient blood supply. In certainembodiments, the patterned biomaterial of the present disclosure can beimplanted in a region of a subject that requires an increase in bloodflow. For example, the patterned biomaterial can be implanted in and/ornear an ischemic tissue. In certain embodiments, the patternedbiomaterial can be implanted to treat cardiac ischemia. The patternedbiomaterial can be implanted to revascularize from healthy coronarycirculation or neighboring non-coronary vasculature.

In certain embodiments, the patterned biomaterial of the presentdisclosure can be used as a novel adjunct to coronary artery bypassgrafting (CABG) in addressing cardiac ischemia. In certain embodiments,during CABG surgery, a surgeon can apply the patterned biomaterial ofthe present disclosure across regions of incomplete reperfusion. Forexample, the patterned biomaterial can be placed in order torevascularize from healthy coronary circulation or neighboringnon-coronary vasculature (such as circulation from the left internalmammary artery) into the ischemic zone unlikely to be addressed by theCABG procedure.

In certain embodiments, the patterned biomaterial can be used to directneovascularization around a section of an artery subject to reducedblood flow or occlusion. In this case, the patterned biomaterial can beused to promote revascularization of a region of ischemic myocardium inaddition to a CABG procedure.

In many patients that suffer from acute myocardial ischemia and inanother even larger cohort of patients with untreatable coronarydisease, there remain areas of viable heart that do not naturallyrevascularized but can be revascularized by an angiogenic tissuescaffold. In certain embodiments, the patterned biomaterial canpotentially revascularize those inaccessible ischemic zones in thesepatients. The selection of an alternating hydrophobic/hydrophilicarrangement of fibers of the patterned biomaterial can stimulate andspatially direct revascularization by directing blood flow from nearbyunobstructed coronary vasculature to around and beyond a coronaryobstruction leading to micro-perfused distal myocardium to protectcardiomyocytes viability and function.

The patterned biomaterial of the present disclosure can enhanceneovascularization as well as influence vascular architecture throughtwo potential mechanisms. The patterned biomaterial can be incorporatedinto existing capillary beds to increase blood flow. Second, thepatterned biomaterial can deliver extracellular matrix constituents andsecrete growth factors into tissue thereby providing a microenvironmentthat promotes angiogenesis.

The patterned biomaterials disclosed herein are meshes comprisingpolymeric nanofibers. The hydrophilic and hydrophobic nanofibers areused to construct the mesh, such that the hydrophilic and hydrophobicnanofibers are disposed in alternating patterns, thereby affecting thesurface energy of the matrix and affect cell growth and healing. In allof the embodiments disclosed throughout, the hydrophobic fibers may be ahydrophobic polymer, including but not limited to polypropylene,polycaprolactone, and polylactic acid. Hydrophilic fibers may behydrophilic polymers, including but not limited to polyether urethanes,polyester urethanes, or polyhyaluronic acid.

The patterned biomaterial of the present disclosure is capable ofenhancing neovascularization by spatially guiding the invading sproutsof an angiogenic capillary network upon implantation, withoutincorporation into the nascent vessels. The patterned biomaterial of thepresent disclosure can be used in conjunction with various types ofengineered tissue constructs to aid in the vascularization of ischemictissue.

In certain embodiments, the patterned biomaterials of the presentdisclosure can be useful in other applications in which it would bebeneficial to have an engineered material to aid in spatially guidingthe direction of host cell and tissue invasion. Such applications caninclude, but are not limited to, nerve regeneration. In certainembodiments, the patterned biomaterial can be seeded with a heterotypiccell suspension. For example, for nerve regeneration applications, thecell suspension can include neurons, neuronal stem cells, or cells thatare associated with supporting neuronal function, or a combinationthereof. In certain embodiments, the patterned biomaterial can be usedat a site of tissue damage, e.g., neuronal tissue damage.

In certain embodiments, the patterned biomaterial of the presentdisclosure can allow for maintenance of the viability and properfunction of a surgical repair site. For example, the patternedbiomaterial can allow for maintenance of the viability and properfunction of muscle tissue surrounding a hernia repair.

In certain embodiments, the patterned biomaterial of the presentdisclosure can enhance wound healing. In certain embodiments, thepatterned biomaterials can be useful in the treatment of chronic woundssuch as, for example, diabetic foot ulcers. Additionally, the patternedbiomaterial of the present disclosure can be useful in the treatment ofwounds sustained during military combat. In certain embodiments, thepatterned biomaterial can be implanted in a subject to treat peripheralvascular disease, diabetic wounds, and clinical ischemia.

In certain embodiments, the patterned biomaterial of the presentdisclosure can be used to enhance repair of various tissues. Examples oftissues that can be treated by the patterned biomaterial of the presentdisclosure includes, but is not limited to, skeletal muscle tissue,skin, fat tissue, bone, cardiac tissue, pancreatic tissue, liver tissue,lung tissue, kidney tissue, intestinal tissue, esophageal tissue,stomach tissue, nerve tissue, spinal tissue, and brain tissue.

In certain embodiments, a method of vascularizing a tissue of a subjectincludes providing a patterned biomaterial comprising endothelial cellsorganized along lines and implanting the patterned biomaterial into atissue of the subject, wherein the biomaterial promotes increasedvascularity and perfusion in the subject.

To facilitate a better understanding of the present disclosure, thefollowing examples of certain aspects of some embodiments are given. Inno way should the following examples be read to limit, or define, theentire scope of the disclosure.

EXAMPLES Example 1: Polymer

Polymers suitable for constructing patterned biomaterials of the presentdisclosure are preferably absorbable in situ. Polyesterurethanes arepolyurethanes copolymerized with a lactide diol. Lactide diol wasprepared using the following materials: 1,6-Hexanediol (Acros), Toluene(Acros) D,L-Lactide (SAFC), L,L-Lactide (Aldrich), Tin-ethylhexanoate(Sigma Aldrich), Chloroform (Sigma Aldrich), Diethylether (Sussmann).

This procedure is to be performed in closed vessels purged continuouslywith cryogenically distilled (dry) argon or nitrogen.

Thirty (30) grams of 1,6-hexanediol is to be placed in 600 ml of toluenein a graduated 2 Liter flat bottom flask equipped with a magnetic stirrod. The flask is to be capped with a 2-hole stopper, one hole equippedwith an input conduit and the other hole equipped with an output conduitconnected to an oil trap (to prevent back flow of water vapor). Theinput conduit is to be connected to the nitrogen source and nitrogenflowed at approximately 5 Liters per hour. The flask is to be placed ona magnetic stirrer/hot top combination.

The toluene solution is to be stirred while raising the solutiontemperature to 70° C., and thereafter in 10° C. increments until thehexanediol is completely dissolved. Upon dissolution, the solutionvolume is to be noted. Temperature and nitrogen flow is to be continueduntil the solution volume drops by 150 ml. Temperature can be raised to130° C. to facilitate toluene vaporization.

A sample of the solution is to be retrieved by syringe (to avoid contactwith humid air), and the toluene removed by vacuum evaporation. A KarlFischer water content measurement is to be performed on the solidhexanediol. The above distillation procedure is to be continued untilthe water content is <300 ppm H₂O by weight. The solution is to becooled and stored under nitrogen.

Using the above setup, 150 grams of D,L-lactide and 150 grams ofL,L-lactide are to be dissolved in 1750 ml of toluene by heating to 115°C., while stirring under nitrogen flow. Upon dissolution the solutionvolume is to be noted and the temperature is to be raised to 130° C. Thenitrogen flow is to be continued until 400 ml of toluene is removed.

A sample of the solution is to be retrieved by syringe (to avoid contactwith humid air), and the toluene removed by vacuum evaporation. A KarlFischer water content measurement is to be performed on the solidhexanediol.

The above distillation procedure is to be continued until the watercontent is <300 ppm H₂O by weight. The solution is to be cooled andstored under nitrogen.

Weigh an appropriately sized flask (4 L). Note flask weight, preferablythe weight includes a closure or the stopper with dosed conduitsdisconnected. The hexanediol and lactide solutions are to be combined inthe weighed flask, connected to nitrogen flow and stirred. The combinedsolution is to be heated in 10° C. increments to 70° C.

After 15 minutes, 600 mg of tin ethylhexanoate is to be added drop-wiseusing a ⋅1 cc syringe, while stirring vigorously. The temperature of thesolution is to be raised to 120° C. in 10° C. increments. [If atemperature-controlled heating mantle is used, the temperature rise willbe sufficiently slow that the 10° C. heating increment can be ignored.]

Turn off the nitrogen flow while keeping conduits connected such thatthe solution volume is closed from contact with air. While stirring andheating, react for 5 hours. Add an additional 400 mg of tinethylhexanoate. Flush with nitrogen. Continue for an additional 3 hours.Add an additional 400 mg of tin ethylhexanoate. Flush with nitrogen.Continue for an additional 11 hours at 120° C. Reduce solutiontemperature to 70° C. Connect the output port of the oil trap to avacuum source. Stop stirring and heat until toluene is removed.

Discontinue vacuum. Add 800 ml of dry chloroform flush with nitrogen,stir at 70° C. until the solid is completely dissolved. The resultingturbid solution is to be filtered using a 0.2-micron PTFE filter. Removethe solvent from the filtrate under vacuum.

A sample of the dried solid is to be measured for water content usingKarl-Fischer. The water content is to be <300 ppm. If not within thisspecification, the solid can be dried by chloroform distillation.

Preparation of Polyesterurethane

Polyesterurethane is prepared from the following materials: IPDI(isophorone diisocyanate) 202.9 mmol, 1,4-Butanediol 142.8 mmol, Toluene2000 ml, Dibutyltin dilaurate 11.6 mmol, PTMG 2000 (Terathane 2000) 20.1mmol, PLA Diol AP1756 40.3 mmol. All operations are to be performedunder nitrogen and dry solvents. Suggested Equipment: A 2 Liter,four-port graduated glass reactor with central port for introduction ofmotor propelled stir rod is recommended. The stir rod is preferablymulti-tier with angled blades to avoid laminar mixing. The reactor is tobe equipped with a heating mantle fitted with a thermocouple and aprogrammable temperature controller. [Preferably, the mantle has coolingcapability as well, in which a fluid filled mantle is used inconjunction with a circulating control unit.] Preferably the reactionvolume is not exposed to the thermocouple, but rather the thermocoupleis embedded in the heating mantle. Due to the high viscosity of thefinal product and need for rapid and complete mixing, use of a magneticstir rod is discouraged. The two free ports are to be equipped withconduits for delivery and removal of nitrogen. The output port is to beconnected to an oil trap to prevent backflow of water vapor. Ideally theconduits contain valves to provide for transport of the reaction volumewithout exposure to air. The last port, the diagnostic port, is to beused for addition and retrieval of reaction volume. The nitrogenatmosphere should be delivered at partial positive pressure tocompensate for the external stirring means and periodic opening of thediagnostic port. The partial pressure is indicated by the observation ofnitrogen bubbles in the oil trap, and the rate of their creation can beused to set and maintain a reasonable nitrogen flow rate.

Purge the reactor with nitrogen. Add 40.32 grams of PLA diol, obtainedfrom the procedure above and 40.11 grams of Terathane 2000 and 810 ml oftoluene using the above setup. Set the stir rate to 100 cycles perminute. The dissolution is accomplished by heating to 115° C., whilestirring under nitrogen flow. Upon dissolution the solution volume is tobe noted and the temperature is to be raised to 130° C. The nitrogenflow is to be continued until 200 ml of toluene is removed.

Cool the reactor to 15° C. (or room temperature, if the mantle is notequipped with coolant). While stirring, add via the diagnostic port andunder nitrogen flow, 30 ml toluene followed by 45.09 grams of IPDI. Stirfor 30 minutes. Add drop wise, 6.74 ml dibutyltin dilaurate.

Using the diagnostic port, remove a sample of the solution to measurethe % NCO. The % NCO can be measured using dibutylamine back titration.By this method, it is traditional to take at least 3 NCO measurements,or you may do so until a desired standard deviation is obtained.

Raise the temperature of the reactor to 75° C. React the mixture undernitrogen flow for 4 hours at 75° C. Take an NCO. React for another 1hour, take an NCO. If the NCO at 5 hours is less than 95% of themeasurement at 4 hours, continue to react for 1-hour durations until theNCO change is less than 5% between consecutive measurements.

Using the setup of the preparation of the PLA diol, dissolve 12.872 g ofbutanediol in 230 ml of dry toluene. Dissolution is accomplished byheating to 75° C.

Add the butanediol solution to the reactor. React the mixture undernitrogen flow for 9 hours at 75° C. Take an NCO. React for another 1hour, take an NCO. If the NCO at 10 hours is less than 95% of themeasurement at 9 hours, continue to react for 1 hour durations until theNCO change is less than 5% between consecutive measurements.

During the course of this procedure, toluene may be added to reduce theviscosity of the reactant and improve mixing. Considerable torque candevelop during this reaction. When the NCO has stabilized [this shouldbe reproducible from batch to batch, if not water is entering thesystem], decant the reaction volume to a vacuum chamber. This is easierperformed if the reaction volume is still hot. Apply vacuum and removethe toluene, and the resulting solid is to be dissolved in 1000 ml THF.The polymer is then precipitated in 15 L of pentane, filtered, washedwith pentane and dried under vacuum at 50° C. n-Pentane can be obtainedfrom Acres and used after re-distillation, and THF (also from Acros) wasused as received.

The resulting polyesterurethane has a melt temperature of 132° C. and issoluble in most solvents, for example toluene and acetone.

Example 2: Bioactive

All of the synthesis that is detailed below are to be performed in ahermetically sealed glass reactor equipped with a stir rodtemperature-controlled jacket. The head space of the reactor is to becontinuously flushed with dry nitrogen unless otherwise specified.

Example 2a: Preparation of a Polyester diisocyanate

In this example a castor-derived hydroxyl-terminated ricinoleatederivative is used as the diol. One equivalent of polycin D-265 (212 g)is combined with 2 equivalent of toluene diisocyanate (174 g) at roomtemperature (22° C.). The mixture is stirred at 100 revolutions perminute and the temperature monitored. The mixture will begin to heat upby exothermic reaction and no heat is to be applied to the reactor untilthe temperature in the reactor ceases to rise. Then the mixturetemperature should be increased in 5° C. increments per ½ hour until themixture reaches 60° C. The reaction should be continued until the %NCO=10.9%. The target % NCO is reached when every hydroxyl group in themixture is reacted with an NCO group. Ideally, the result is a singlediol endcapped with two diisocyanates. This outcome can be enhanced byslow addition of the diol to the diisocyanate. The addition should be in⋅10 g increments, added when the exotherm from the previous addition hasceased. However, chain extended variations of the above ideal outcomeare useful, their primary disadvantage being that the product isslightly higher in viscosity. The ideal % NCO is calculated by dividingthe weight of the functional isocyanate groups (2×42 Dalton) per productmolecule by the total weight of the product molecule (424 Dalton+2×174Dalton) yielding approximately 10.9%.

Alternatively, a lower molecular weight diol may be used, such aspolycin D-290 where 1 equivalent of polycin D-290 is 193 g and thetarget % NCO is 84/(386+348)=11.4%.

Alternatively, a higher molecular weight diol may be used, such aspolycin D-140 where 1 equivalent of polycin D-140 is 400 g and thetarget % NCO is 84/(800+348)=7.3%.

All polycin diols are available from Performance Materials (Greensboro,NC) and toluene diisocyanate is available from Sigma-Aldrich (Milwaukee,Wisc.).

Example 2b: Preparation of a Polyether diisocyanate

In this example a polyether hydroxyl-terminated copolymer of 75%ethylene oxide and 35% propylene oxide is used as the diol. Oneequivalent of UCON 75-H-450 (490 g) is combined with 2 equivalent oftoluene diisocyanate (174 g) at room temperature (22° C.). The mixtureis stirred at 100 revolutions per minute and the temperature monitored.

The mixture will begin to heat up by exothermic reaction and no heat isto be applied to the reactor until the temperature in the reactor ceasesto rise. Then the mixture temperature should be increased in 5° C.increments per ½ hour until the mixture reaches 60° C. The reactionshould be continued until the % NCO=10.9%. The target % NCO is reachedwhen every hydroxyl group in the mixture is reacted with an NCO group.Ideally, the result is a single diol endcapped with two diisocyanates.This outcome can be enhanced by slow addition of the diol to thediisocyanate. The addition should be in 10 g increments, added when theexotherm from the previous addition has ceased. However, chain extendedvariations of the above ideal outcome are useful, their primarydisadvantage being that the product is slightly higher in viscosity. Theideal % NCO is calculated by dividing the weight of the functionalisocyanate groups (2×42 Dalton) per product molecule by the total weightof the product molecule (980 Dalton+2×174 Dalton) yielding approximately6.3%. Polyether copolymers of ethylene oxide and propylene oxide diolsare available from Dow Chemical (Midland, MI).

Example 2c: Preparation of a Polyester triisocyanate

In this example a castor-derived hydroxyl-terminated ricinoleatederivative is used as the triol. One equivalent of polycin T-400 (141 g)is combined with 2 equivalent of toluene diisocyanate (174 g) at roomtemperature (22° C.). The mixture is stirred at 100 revolutions perminute and the temperature monitored. The mixture will begin to heat upby exothermic reaction and no heat is to be applied to the reactor untilthe temperature in the reactor ceases to rise. Then the mixturetemperature should be increased in 5° C. increments per Y2 hour untilthe mixture reaches 60° C. The reaction should be continued until the %NCO=13.3%. The target % NCO is reached when every hydroxyl group in themixture is reacted with an NCO group. Ideally, the result is a singlediol endcapped with two diisocyanates. This outcome can be enhanced byslow addition of the diol to the diisocyanate. The addition should be in10 g increments, added when the exotherm from the previous addition hasceased. However, chain extended variations of the above ideal outcomeare useful, their primary disadvantage being that the product isslightly higher in viscosity. The ideal % NCO is calculated by dividingthe weight of the functional isocyanate groups (2×42 Dalton) per productmolecule by the total weight of the product molecule (282 Dalton+2×174Dalton) yielding approximately 13.3%.

The above reaction will yield a viscous product. A less viscous productcan be obtained by adding propylene carbonate to the initial mixture.Additions up to 100% by weight of propylene carbonate are useful.Adjustment to the target NCO of the mixture must be performed usingstandard methods, or the propylene carbonate may be added after reachingthe target % NCO. Propylene carbonate is available from Sigma-Aldrich(Milwaukee, Wisc.).

Example 2d: Preparation of a Polyether triisocyanate

In this example a polyether hydroxyl-terminated copolymer of 75%ethylene oxide and 35% propylene oxide is used as the trio!. Oneequivalent of Multranol 9199 (3066 g) is combined with 3 equivalent oftoluene diisocyanate (261 g) at room temperature (22° C.). The mixtureis stirred at 100 revolutions per minute and the temperature monitored.The mixture will begin to heat up by exothermic reaction and no heat isto be applied to the reactor until the temperature in the reactor ceasesto rise. Then the mixture temperature should be increased in 5° C.increments per ½ hour until the mixture reaches 60° C. The reactionshould be continued until the % NCO=1.3%. The target % NCO is reachedwhen every hydroxyl group in the mixture is reacted with an NCO group.Ideally, the result is a single diol endcapped with two diisocyanates.This outcome can be enhanced by slow addition of the diol to thediisocyanate. The addition should be in 10 g increments, added when theexotherm from the previous addition has ceased. However, chain extendedvariations of the above ideal outcome are useful, their primarydisadvantage being that the product is slightly higher in viscosity. Theideal % NCO is calculated by dividing the weight of the functionalisocyanate groups (3×42 Dalton) per product molecule by the total weightof the product molecule (9199 Dalton+3×174 Dalton) yieldingapproximately 1.3%. Multranol 9199 is available from Bayer (Pittsburg,PA).

Example 2e: Preparation of a polyol triisocyanate from polyol diol

Any of the diisocyanates prepared in Examples 2a and 2b can betrimerized by the addition of a low molecular weight triol such aspolycin T-400 or trimethylolpropane (TMP). In this example TMP is used,but the method is adaptable to any triol. Complete trimerization of thediisocyanates of Example 2a and 2b will result in viscous products.

To yield a lower viscosity product propylene carbonate can be employedor less triol can be used. In the later case, a mixture of diisocyanateand triisocyanate is obtained. In this example the product of Example 2bis used as the polyether diisocyanate. One equivalent of Example 2b (682g) is combined with 0.1 equivalent TMP (44.7 g) at room temperature (22°C.). The mixture is stirred at 100 revolutions per minute and thetemperature monitored. The mixture will begin to heat up by exothermicreaction and no heat is to be applied to the reactor until thetemperature in the reactor ceases to rise. Then the mixture temperatureshould be increased in 5° C. increments per half hour until the mixturereaches 60° C. The reaction should be continued until the % NCO=5.8%.

The target % NCO is reached when every hydroxyl group in the mixture isreacted with an NCO group. The ideal % NCO is calculated by dividing theweight fraction of the functional isocyanate groups 10% (3×42 Dalton)and 90% (2×42) per product molecule by the total weight fraction of theproduct molecule (3×1364 Dalton+⋅134 Dalton)+1364 yielding approximately0.3%+5.5%=5.8%. TMP is available from Sigma-Aldrich (Milwaukee, Wisc.).

Example 2f: Preparation of a Modified Boswellia Extract using thetriisocyanate of Example 2d

The hydroxyl number of Boswellia extract will vary depending onextraction method, species of Boswellia extracted, and even variationswithin species. The goal is to obtain a product with no NCOfunctionality, so all reaction mixtures should be reacted until thefinal % NCO=0

In this example the product of Example 2d is used as the polyethertriisocyanate mixture. One hundred grams of Example 4 is combined with 1g of Boswellia extract at room temperature (22° C.) under 90% nitrogenand 10% nitric oxide atmosphere. The mixture is stirred at 100revolutions per minute and the temperature monitored. The mixture willbegin to heat up by exothermic reaction. When the temperature ceases torise, a % NCO reading is taken. If % NCO>0 than an additional 1 g ofBoswellia extract is to be added. By a series of Boswellia addition onecalculates the change in % NCO as a function of 1 g additions ofBoswellia extract, a linear plot is obtained from which the total amountof Boswellia extract addition necessary to bring the % NCO to zero isobtained. This amount of Boswellia extract is added to the mixture andthe mixture is reacted so that % NCO=0 is obtained.

Example 2g: Preparation of a Modified Boswellia Extract using thetriisocyanate/diisocyanate of Example 2e

The hydroxyl number of Boswellia extract will vary depending onextraction method, species of Boswellia extracted, and even variationswithin species. The goal is to obtain a product with no NCOfunctionality, so all reaction mixtures should be reacted until thefinal % NCO=0.

In this example the product of Example 2e is used as the polyetherdiisocyanate/triisocyanate mixture. One hundred grams of Example 2e iscombined with 1 g of Boswellia extract at room temperature (22° C.)under 90% nitrogen and 10% nitric oxide atmosphere. The mixture isstirred at 100 revolutions per minute and the temperature monitored. Themixture will begin to heat up by exothermic reaction. When thetemperature ceases to rise, a % NCO reading is taken. If % NCO>0 then anadditional 1 g of Boswellia extract is to be added. By a series ofBoswellia addition one calculates the change in % NCO as a function of 1g additions of Boswellia extract, a linear plot is obtained from whichthe total amount of Boswellia extract addition necessary to bring the %NCO to zero is obtained. This amount of Boswellia extract is added tothe mixture and the mixture is reacted so that % NCO=0 is obtained.

Example 2h: Preparation of a highly-Branched Modified Boswellia Extractwith Absorbable Links

Diol and triol can be combined to form a multi-branch polymer. In thisinstance, the Multranol 9199 triol is chain extended with polycin D-265diol. The diisocyanate form of Example 2 is useful in chain extendingthe triisocyanate form of Example 4. We wish to have on average 2diisocyanates for every 3 triisocyanates, which forms a 5-armedisocyanate.

In this example 0.09 equivalents (292 g) of Example 2d is mixed with0.04 equivalents (26.6 g) of Example 2b. The triisocyanates of Example2d and diisocyanates of Example 2b are chain extended with 0.08equivalents lysine diamine to form a 5-armed isocyanate. One hundredgrams of this reaction product is combined with 1 g of Boswellia extractat room temperature (22° C.) under 90% nitrogen and 10% nitric oxideatmosphere. The mixture is stirred at 100 revolutions per minute and thetemperature monitored. The mixture will begin to heat up by exothermicreaction. When the temperature ceases to rise, a % NCO reading is taken.If % NCO>0 than an additional 1 g of Boswellia extract is to be added.By a series of Boswellia addition one calculates the change in % NCO asa function of 1 g additions of Boswellia extract, a linear plot isobtained from which the total amount of Boswellia extract additionnecessary to bring the % NCO to zero is obtained. This amount ofBoswellia extract is added to the mixture and the mixture is reacted sothat % NCO=0 is obtained. Lysine diamine is available from Sigma-Aldrich(Milwaukee, Wisc.),

Example 3: Surface Texture

A patterned biomaterial of the present disclosure can be given a surfacetexture by laying down polymer on a textured surface during the printprocess when the polymer is liquid. Alternatively, the finishedbiomaterial can be impressed on a heated mold causing the surface of thebiomaterial to melt into the mold texture. Textures of particularinterest are those with features characterized by multiple size scales,for example, features of approximate size 1 micron, 10 microns and 100microns. Preferably, the features are superimposed such that the 1micron features reside on the 10 micron features, and the 10 micronfeatures reside on the 100 micron features.

Preferred patterns resemble the surface of rose petals of genus Rosa.Referring to FIG. 1A, depicts a cross sectional view of a rose patternmimic 100 is comprised of a large-scale pattern 102, the surf ace ofwhich is approximately a sinusoid or similar undulating surface ofamplitude 104 and pitch 105. The amplitude can range from 50 microns to250 microns. Preferably, the amplitude is 50 microns, more preferably200 microns, or most preferably 250 microns. The pitch can range from 50microns to 250 microns. Preferably, the pitch is 50 microns, morepreferably 200 microns, or most preferably 250 microns. A medium scalepattern is comprised of cylindrical pillars 106 of diameter 108.Diameter 108 can range from 5 to 50 microns, 10 to 50 microns, 10 to 30microns or 5 to 20 microns. In some embodiments, the diameter is about30 microns, more preferably about 20 microns, most preferably about 10microns. The height 110 of the pillars is approximately 20 to 50microns.

FIG. 1 b represents a top view of large scale structure 102. The pillars106 are distributed on a square grid 111 of 30 to 100 micron centers. Insome embodiments, the centers are about 30 to 80 microns, 40 to 70microns or about 50 microns.

FIG. 1C depicts an expanded view of pillar 106 comprising smallerpillars 118. FIG. 1D depicts an expanded top view of pillar 116 havingsmaller pillars 118 disposed thereon and ridges 112. Ridges 112 can havea height 114 of 0.5 to 10 microns, 0.5 to 5 microns, about 5 microns, orabout 1 micron. The ridges 112 are spaced apart by distance 113 of 1 to10 microns, or about 5 microns, and approximately parallel along theirlength. The tops 116 of the pillars 106 may be populated with smallerpillars 118 of diameter 120 0.5 to 5 micron or about 1 micron and height122 of 0.5 to 1 micron or approximately 1 micron. These smaller pillars118 are distributed on a square grid 124 of 1 to 10 micron centers, orabout 5 micron centers. This rose mimic pattern can in some embodimentsbe impressed directly on the fibers of the patterned biomaterial orsheets with this pattern can be affixed to portions of the patternedbiomaterial.

Example 4: Patterned Biomaterial

Using a print electrospin device containing two print heads, one printhead is loaded with a hydrophilic polyesterurethane and the other printhead is loaded with a hydrophobic polylactic acid polymer. The printheads are heated such that the polymers are in a liquid state.

Referring to FIG. 2 , the electrospin device is loaded with a computeraided design to form a square mesh 200 design. The square mesh iscomprised of fibers 202 arranged in a square pattern where the voids 204provide an open porous structure suitable for seeding or ingrowth ofcells. The patterned biomaterial is comprised of fibers 202 laid down onfirst and second orthogonal axes to form alternating layers 208 and 210.In one embodiment, the fibers forming layers 208 are hydrophobic and thefibers forming layers 210 are hydrophilic. The height 206 is determinedby number of fibers 202 layered or stacked atop one another. Thehydrophobic fibers may be a hydrophobic polymer, including but notlimited to polypropylene, polycaprolactone, and polylactic acid.Hydrophilic fibers may be hydrophilic polymers, including but notlimited to polyether urethanes, polyester urethanes, or polyhyaluronicacid. The fibers are, in some embodiments, nanofibers ranging from about100 nm to about 5 microns, or about 100 nm to about 1 micron, or about100 nm to about 500 nm.

In another embodiment, the fibers that form layers 208 are alternatelyhydrophilic and hydrophobic. In this embodiment, the fibers forminglayers 210 are alternately hydrophilic and hydrophobic. Consequently,the juxtaposed lines of layer 208, for example line 212, and layer 210,for example line 214, are either both hydrophilic or both hydrophobic,and are stacked in alternating fashion. The resulting patternedbiomaterial is useful for promoting neovascularization at a wound repairsite. In some embodiments, the voids 204 have a diameter ranging from 25microns to 5 mm, 25 microns to 1 mm, 25 microns to 500 microns or 50microns to 200 microns.

Example 5: Patterned Biomaterial

In some embodiments, the patterned biomaterial is maximally flexible.For example, a chain mail design. In this embodiment, two polymers aredelivered in solution. A first polymer is dissolved in a first solvent.A second polymer is dissolved in a second solvent. The second polymer isnot soluble in the first solvent. The first polymer is not soluble inthe second solvent. Using a print electrospin device, where the polymerstream can be interrupted, a matrix of line segments are laid down.

Referring to FIG. 3A-3C, a chain mail patterned biomaterial 300 isconstructed by first laying down a square grid 302 of line segments 304,as seen in FIG. 3A. The line segments may have a length of 2000 microns,in some embodiments. The line segments are spaced colinearly on 4000micron centers 306 and spaced laterally 308 by 4000 microns. The lines304 comprise a first polymer. After grid 302 has solidified, a secondlayer 312 comprised of circles 314 is laid down, as depicted in FIG. 3B.Circles 314 may have a diameter of 3000 microns 315 and are centered atintersection points 316. The circles comprise second polymer. The firstpolymer of 304 is insoluble in the solvent of second polymer 314, suchthat line segments 304 do not adhere to circles 314. After second layer312 has solidified third layer 320 is laid down, as depicted in FIG. 3C.Third layer is comprised of the first polymer. Line segments 322 arelaid down as depicted, where line segment ends 324 are the only pointson line segment 322 in contact with line segments 304. When line segment322 contacts line segment 304 the solvent in 322 partially dissolvessegment 304 such that segments 322 and 304 form a continuous loop. Afterthird layer 320 solidifies, the result is a chain mail structure whereinloops of the first polymer are interlocked with loops of the secondpolymer, and first polymer loops slidably translate and rotate insidesecond polymer loops. In some embodiments, the loops have a diameterranging from 50 microns to 5 mm, 50 microns to 2 mm, 50 microns to 1 mm,100 microns to 2 mm, 100 microns to 1 mm or 500 microns to 1 mm.

Example 6: Patterned Biomaterial

In some embodiments, a composite sheet and mesh construct is useful invarious soft tissue repair applications. In this embodiment, the sheetcomponent is polyesterurethane and the mesh component is polylacticacid, wherein the melt temperature of the polylactic acid is lower thanthe melt temperature of the polyesterurethane. The polyesterurethane isimprinted with a rose petal pattern designed to prevent migration of theimplant and promote endothelial cell motility. In other embodiments, thesheet can be a polylactic acid, a polycaprolactone, polypropylene,polyether urethanes, or polyhyaluronic acid.

Referring to FIG. 4 , a composite sheet and mesh patterned biomaterial400 is depicted. A first layer of lines 402 of polylactic acid is laiddown on 8000 micron centers 404 and spaced apart 406 by 8000 microns.Each line 402 is 3500 microns long 407. A sheet of polyesterurethane 408is comprised of 1000 micron diameter holes 410 spaced 4000 microns apart412. The sheet 408 is registered with respect to line grid 402 such thatthe space 414 between line segments 402 is centered between sheet holes410 in two dimensions 416 and 418. After registration, a second layer ofpolylactic acid lines 420 is laid down. At each endpoint 422 of eachline 424 is started a line 420 that terminates at a second endpoint 426of an adjacent line 428. The temperature of the polylactic acid issufficient to melt-bond endpoints 422 and 426 to line 420, such thatwhen solid, polylactic acid lines weave above and below thepolyesterurethane sheet. The hydrophobic polylactic acid strands promotefibroblasts through the polyesterurethane sheet whereas the texturedsurface of the polyesterurethane sheet promotes endothelialization andneovascularization in the plane of the polyesterurethane sheet.

Example 7: Patterned Biomaterial

In some embodiments a three-dimensional mesh is desired. Athree-dimensional mesh can be printed on a two-dimensional surface.Square matrices of lines can be stacked without adhesion by laying downa first square matrix of melt polymer, cooling this layer sufficientlysuch that when a second layer of square matrix is laid down over thefirst, the second layer solidifies prior to the first layer heatingsufficiently to melt. Non-adhering layers stacked in this way can beconnected together by running a diagonal line of polymer melt such thatthe diagonal line melts into the vertices of the stacked squarematrices.

Referring to FIG. 5 , a three-dimensional mesh 500 is depicted. Firstsquare matrix 502 is laid down and solidified and cooled to 20centigrade below the melting point. Then a second square matrix 504 islaid down such that the intersection points 506 of matrix 504 arediagonally offset distance 508 from intersection points 510 of matrix502. An arbitrary number of matrices of type 502 can be laid downwithout adherence between them. In a final step, polymer is laid down indiagonal lines 512. The polymer bonds at the intersection points 506 and510 joining matrices 504 and 502, respectively. The patternedbiomaterial can be expanded in a third dimension 514 by suspending thebiomaterial from diagonal lines 512 and heating sufficient to relax thepolymers without melting them.

Referring now to FIG. 6 , an exemplary mesh device is depicted. A humantissue with defect and an implant modification 600 comprises a humantissue 610. 610 can be bone, muscle, or any structure in the body thatcan be repaired endogenously by cellular infiltration. 610 contains adefect 620. Microtextured haptotaxic implant is in this instance a mesh630, the filaments 640 of the mesh are selected to promote functionalrepair of the defect.

FIG. 7 is an image of a mesh 200 according to the present disclosurehaving alternating hydrophobic and hydrophilic fibers. In someembodiments, the fibers alternate as they are stacked, while in otherembodiments, the fibers alternate adjacent to one another.

FIG. 8 is another image of an embodiment of a mesh 200 according to thepresent disclosure.

FIG. 9 is further matrix pattern 900, in which fibers are arranged in aseries of stacked triangles 902, which are centered about a point 904.The triangles comprises polymer nanofibers and can alternate betweenhydrophilic and hydrophobic fibers.

FIG. 10 depicts a matrix having an alternative rose petal mimic 1000having fibers disposed in a hierarchical grid pattern. The grid isformed from macro layers of 50 microns to 1 mm (1002), meso layers of 1micron to 50 micron Cl004) and fine layers of less than 1 micron (1006).In some embodiments, the macro scale ranges from 50 microns to 500microns, or 100 microns to 500 microns. In some embodiments, the mesoscale ranges from 10 microns to 30 microns or 5 microns to 25 microns.In some embodiments, the fine scale ranges from 0.1 micron to 1 micron,1.1 micron to 0.75 micron or 0.25 micron to 0.75 micron. The surfaceenergy of the matrix thus is varied on at least three spatial scalescomprising 1) a macro scale ranging from 50 micron to 1 mm obtained byforming layers of polymers of different surface energy, 2) a meso scaleranging from 1 micron to 50 micron obtained by placing a surface patternon the polymers, and 3) a fine-scale of less than 1 micron obtained byvariation of molecular structure.

What is claimed is:
 1. A three-dimensional matrix deposited viaelectrospinning for inducing tissue regeneration comprising: at leastthree successive layers of at least two biocompatible polymers, a firstlayer of the at least three successive layers comprising a substrate, asecond layer and a third layer of the at least three successive layersarranged in a stacked pattern to form a plurality of walls with an openvoid disposed between the plurality of walls, the second layer and thirdlayer deposited via electrospinning onto the substrate, and wherein thesubstrate comprises a microstructure pattern disposed thereon, themicrostructure pattern having a first surface energy and second surfaceenergy capable of electrostatically directing the deposition location ofthe second layer and the third layer.
 2. The three-dimensional matrix ofclaim 1, wherein the microstructure pattern comprises a plurality ofmicrostructures having a first microstructure of the plurality ofmicrostructures including the first surface energy and a secondmicrostructure of the plurality of microstructures having the secondsurface energy wherein the first surface energy is different than thesecond surface energy.
 3. The three-dimensional matrix of claim 2,wherein the first microstructure is disposed hierarchically on thesecond microstructure.
 4. The three-dimensional matrix of claim 1,wherein the deposition of the second layer and the third layer isdependent at least in part by the location of the microstructure patternon the substrate.
 5. The three-dimensional matrix of claim 1, whereinthe microstructure pattern is capable of generating an adhesive forcewith respect to a target surface.
 6. The three-dimensional matrix ofclaim 1, wherein the at least three successive layers of at least twobiocompatible polymers is capable of generating an adhesive force withrespect to a target surface.
 7. The three-dimensional matrix of claim 1,wherein the matrix is capable of generating a tissue scaffolding effectto promote tissue regeneration.
 8. The three-dimensional matrix of claim1, wherein in an implanted state, at least a first portion is capable ofadhering to biological tissue and at least a second portion is capableof promoting tissue regeneration in a defect of the biological tissue.9. A three-dimensional matrix deposited via electrospinning for inducingtissue regeneration comprising: at least three successive layers of atleast two biocompatible polymers, a first layer of the at least threesuccessive layers comprising a substrate, a second layer and a thirdlayer of the at least three successive layers arranged in a stackedpattern to form a plurality of walls with an open void disposed betweenthe plurality of walls, the second layer and third layer deposited viaelectrospinning onto the substrate, wherein the substrate comprises amicrostructure pattern disposed thereon, the microstructure patternhaving a first surface energy and second surface energy capable ofelectrostatically directing the deposition location of the second layerand the third layer, and wherein at least one portion of the matrix isbioasorbable.
 10. The three-dimensional matrix of claim 9, wherein thesubstrate and microstructure pattern are bioabsorable.
 11. Thethree-dimensional matrix of claim 9, wherein the second layer and thirdlayer are configured to form a mesh such that the mesh isnon-absorbable.
 12. The three-dimensional matrix of claim 11, whereinthe mesh is capable of providing a reinforcement structure to abiological tissue when implanted.
 13. The three-dimensional matrix ofclaim 9, wherein the microstructure pattern comprises a plurality ofmicrostructures having a first microstructure of the plurality ofmicrostructures including the first surface energy and a secondmicrostructure of the plurality of microstructures having the secondsurface energy wherein the first surface energy is different than thesecond surface energy.
 14. The three-dimensional matrix of claim 13,wherein the first microstructure is disposed hierarchically on thesecond microstructure.
 15. The three-dimensional matrix of claim 9,wherein the second layer and the third layer are deposited viaelectrospinning onto the substrate.
 16. The three-dimensional matrix ofclaim 9, wherein the microstructure pattern is capable of generating anadhesive force with respect to a target surface.
 17. Thethree-dimensional matrix of claim 9, wherein the at least threesuccessive layers of at least two biocompatible polymers is capable ofgenerating an adhesive force with respect to a target surface.
 18. Thethree-dimensional matrix of claim 9, wherein the matrix is capable ofgenerating a tissue scaffolding effect to promote tissue regeneration.